Abstract
Marine molluscs are rich in biologically active natural products that provide new potential sources of anti-inflammatory agents. Here we used bioassay guided fractionation of extracts from the muricid Dicathais orbita to identify brominated indoles with anti-inflammatory activity, based on the inhibition of nitric oxide (NO) and tumour necrosis factor α (TNFα) in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages and prostaglandin E2 (PGE2) in calcium ionophore-stimulated 3T3 ccl-92 fibroblasts. Muricid brominated indoles were then compared to a range of synthetic indoles to determine structure-activity relationships. Both hypobranchial gland and egg extracts inhibited the production of NO significantly with IC50 of 30.8 and 40 μg/mL, respectively. The hypobranchial gland extract also inhibited the production of TNFα and PGE2 with IC50 of 43.03 µg/mL and 34.24 µg/mL, respectively. The purified mono-brominated indole and isatin compounds showed significant inhibitory activity against NO, TNFα, and PGE2, and were more active than dimer indoles and non-brominated isatin. The position of the bromine atom on the isatin benzene ring significantly affected the activity, with 5Br > 6Br > 7Br. The mode of action for the active hypobranchial gland extract, 6-bromoindole, and 6-bromoisatin was further tested by the assessment of the translocation of nuclear factor kappa B (NFκB) in LPS-stimulated RAW264.7 mouse macrophage. The extract (40 µg/mL) significantly inhibited the translocation of NFκB in the LPS-stimulated RAW264.7 macrophages by 48.2%, whereas 40 µg/mL of 6-bromoindole and 6-bromoistain caused a 60.7% and 63.7% reduction in NFκB, respectively. These results identify simple brominated indoles as useful anti-inflammatory drug leads and support the development of extracts from the Australian muricid D. orbita, as a new potential natural remedy for the treatment of inflammation.
Highlights
Inflammation is a complex mechanism involving the activation and deactivation of immune cells in response to stimuli and tightly regulated signalling pathways
This study aims to investigate the inhibition of nitric oxide (NO), tumour necrosis factor α (TNFα), prostaglandin E2 (PGE2), and NFκB translocation by extracts and brominated indoles isolated from hypobranchial gland and egg masses of the Muricidae mollusc Dicathais orbita
The methanol extract from the hypobranchial gland (Figure 1d) was dominated by the precursor tyrindoxyl sulfate and contained some murexine
Summary
Inflammation is a complex mechanism involving the activation and deactivation of immune cells in response to stimuli and tightly regulated signalling pathways. Macrophages play a key role in initiating and maintaining the inflammatory response, and are activated by pathogen associated molecules and cytokines, which stimulate pro-inflammatory signalling pathways [2]. Mar. Drugs 2017, 15, 133 kappa B (NFκB) has long been considered a prototypical pro-inflammatory signalling molecule, activated by pro-inflammatory cytokines such as tumour necrosis factor α (TNFα) in the presence of viruses, genotoxic agents, or when stimulated by microbial constituents, including lipopolysaccharides (LPS) from bacterial cell walls [3]. NFκB translocates to the nucleus, where it regulates the expression of a variety of transcription factors and co-factors, leading to the expression of pro-inflammatory enzymes like cyclooxygenase 2 (COX-2)
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